PIVKA-II (Protein induced by vitamin K absence-II) is a glycoprotein having a structure similar to prothrombin, which is involved in blood coagulation. Prothrombin is a protein with 622 residues, and contains γ-carboxyglutamic acid (Gla) residues generated by γ-carboxylation of 10 glutamic acid (Glu) residues in the vicinity of the N-terminus. It is known that, during production of the prothrombin in the body, incomplete γ-carboxylation may occur due to lack of vitamin K, hepatic insufficiency, administration of a vitamin K antagonist, liver cell damage or the like, and glycoproteins in which all or part of the 10 residues are Glu residues may be found in blood. These proteins are PIVKA-II, which is also called abnormal prothrombin. Recently, detection of PIVKA-II at high concentration in plasma of patients with hepatocellular carcinoma has been reported, and PIVKA-II has begun to be used as a marker for monitoring diagnosis of hepatocellular carcinoma.
As a method for specifically detecting PIVKA-II in a sample, an immunoassay using both a monoclonal antibody that specifically recognizes PIVKA-II and a polyclonal antibody against prothrombin, one of which is an immobilized antibody and the other of which is a labeled antibody, has been reported (Patent Document 1).
In cases where a test substance in a blood-derived sample is measured, serum or plasma is mainly used. Depending on properties of the test substance, the assay system and the like, different measured values for the test substance may be obtained between the serum sample and the plasma sample in paired serum and plasma samples obtained from the same subject. That is, in some cases, the serum-plasma correlation is low and only one of the serum and plasma samples is applicable to the assay. This is especially problematic in cases where two test substances are measured at the same time. For example, in cases where two test substances are measured at the same time and one of the test substances can be measured only with serum, we may collect only a serum sample from the patient if the other test substance can be measured also with serum. However, in cases where the other test substance can be measured only with a plasma sample, both serum and plasma samples need to be collected from the patient. This increases the labor of the sample collection and treatment, as well as the burden on the patient. Thus, establishment of an assay system with high serum-plasma correlation has been demanded.
It is known that, also in measurement of PIVKA-II by ELISA using a monoclonal antibody that specifically recognizes PIVKA-II as an immobilized antibody and a polyclonal antibody against prothrombin as a secondary antibody, thrombin-reactive antibodies contained in the secondary antibody adversely affect the assay system for serum samples, resulting in unstable measured values (Patent Document 2). In order to solve this problem, Patent Document 2 reports that stable measurement of PIVKA-II can be attained even with a serum sample by using as a secondary antibody an antibody that does not react with human thrombin but specifically reacts with human prothrombin. However, in the method described in Patent Document 2, in order to remove antibodies against thrombin from the polyclonal antibody against human prothrombin, it is necessary to use a human prothrombin affinity column to obtain antibodies reactive with prothrombin and then to carry out dialysis, and further thereafter to use a human thrombin affinity column to obtain antibodies that are not reactive with thrombin and then to carry out dialysis. Thus, the antibody purification process is very complicated. Moreover, although the method of Patent Document 2 can also improve the serum-plasma correlation, its further improvement has been demanded. Furthermore, since polyclonal antibodies show variability among lots, monoclonal antibodies are generally more preferred than polyclonal antibodies for strictly securing the specificity. Although Patent Document 2 also describes use, in a PIVKA-II measurement method by ELISA, of a monoclonal antibody which does not react with human thrombin and is specifically reactive with human prothrombin, there is no description on the influence of use of such a monoclonal antibody on serum-plasma correlation, or problems caused by such use.
Patent Document 3 describes an immunoassay using both a monoclonal antibody that specifically recognizes PIVKA-II and a monoclonal antibody against prothrombin, one of which is used as an immobilized antibody and the other of which is used as a labeled antibody. The document also describes use of a mixture of PIVKA-II-specific monoclonal antibodies. However, Patent Document 3 originally does not describe the problem of serum-plasma correlation in the immunoassay system at all, and the influence of the method described in Patent Document 3 on serum-plasma correlation is not clear.